We have previously identified that Her-2/neu transgenic mice crossed with the HLA-A2.1/Kb transgenic mice (A2xneu) are immunotolerant against Her-2/neu/A2.1 immunodominant epitopes. We have also demonstrated that immunization with peptides or Her-2/neu proteins does not provide an effective antitumor response against tumors in A2xneu mice. One of the consequences of crossing the Her-2/neu mice with the HLA-A2.1/Kb mice is that in these animals spontaneous tumors appear when animals are 20-22 months old. Therefore, the A2xneu mouse model provides a unique opportunity to evaluate antitumor immune responses against a self antigen where aging and tolerance are present at the same time. We have demonstrated that aging drastically altered the immune system of old mice. Therefore, it is critical to identify and optimize a vaccination strategy that effectively stimulates an immune response resulting in tumor rejection in young and old tolerant animals. There is plenty of evidence indicating that targeting APCs with different types of adjuvants results in the induction of an antitumor immune response. We compared the antitumor effect of different TLR-ligands (Poly I:C, LPS, flagellin, imiquimod, CpG-ODN) in young and old A2xneu mice. The result indicated that only intratumoral (i.t.) injections of CpG-ODN induced the rejection of tumors in young and old A2xneu mice. Although young and old A2xneu mice could reject the primary tumor after treatment with i.t injections of CpG-ODN, these animals could not develop a protective memory response. Analysis of the tumor microenvironment following i.t injections of CpG-ODN indicated: 1) activation of APCs;2) a pro-inflammatory Th1 type response;3) activation of CD4, CDS T cells and NK cells;and 4) the numbers of T-regs were drastically reduced. Taken together, these results indicate that i.t. injections of CpG-ODN influence the tumor microenvironment that favors the antitumor response. However, we do not yet completely understand how CpG-ODN injections induce the rejection of tumors. The goal of this proposal is to determine the mechanism of action of CpG-ODN in the induction of antitumor responses and, to develop a strategy to generate memory responses in old tolerant hosts. In these studies we will: 1) evaluate and characterize the effect of CpG-ODN on the induction of immune responses in old mice;2) evaluate the relationship between CpG-ODN and T-regs, and the relationship of T-regs in regulating the repertoire and activation of immune responses in old mice;and 3) optimize the use of anti-neu-CpG-ODN to target the CpG-ODN at the tumor site for the treatment of tumors in old mice. Relevance: For the first time our results show that targeting CpG-ODN at the tumor site restores the immune response in old mice. Characterizing the effect of CpG-ODN on the immune system will ultimately lead to the optimization of this therapy. Furthermore, with the generation of the anti-neu-CpG-ODN molecule we can target the CpG-ODN anywhere in the body making it possible to develop a universal vaccine strategy for the treatment of cancer. Overall, the information gained from these studies will reveal strategies for controlling and manipulating the immune system to develop more effective immunotherapies in young and old tolerant hosts.